We seek to understand the molecular responses that occur when skin is exposed to UV radiation in order to devise novel strategies to protect against damage and carcinogenesis. In this proposal we will examine two major risk factors for skin cancer: UV radiation and pigment (melanin) phenotype. Our studies focus on the melanocyte, and its cell surface receptor, the melanocortin-1 receptor (Mc1r), which regulates melanin synthesis. We hypothesize that Mc1r protects melanocytes against UV-mediated mutagenesis and transformation by modulating melanization and recovery from UV-mediated cellular injury. Loss-of-function polymorphisms in Mc1r correlate with fair skin and a high incidence of melanoma, while robust Mc1r function correlates with darker skin and UV resistance. We have developed a novel genetically-matched murine model of human skin as well as a protocol for growing primary melanocytes from these mice. These isogenic melanocytes span eumelanotic, pheomelanotic, and amelanotic melanin composition, will serve a unique and potent tool to delineate the role of Mc1r function in response to UV. Using our unique animal model and primary melanocytes derived thereof, we propose the following aims: Aim 1. Characterize the mechanism of Mc1r-mediated enhancement of nucleotide excision repair (NER); Aim 2. Determine whether Mc1r signaling protects against UV-mediated oxidative damage; and Aim 3. Determine the ability of pharmacologic Mc1r rescue to protect against UV damage. With respect to Aim 1, we will examine the role of Mc1r in the repair of UV-induced photolesions by southwestern and flow cytometric analysis and determine the influence of Mc1r on cellular levels of nucleotide excision repair enzymes by qRT-PCR and Western analysis. We will investigate a molecular link between Mc1r signaling and enhanced repair enzyme levels by examining expression of the NER enzymes basally and in the setting of UV irradiation and by investigating the ability of cAMP-responsive transcription factors downstream of Mc1r signaling (namely Mitf and CREB) to bind to and induce transcription of NER enzyme promoters. In Aim 2 we will study whether pheomelanin, which is produced as a result of low Mc1r function, promotes oxidative damage in melanocytes by complementary measures of oxidative load (DCF-mediated fluorescence flow cytometry, Southwestern blotting, hOGG1-adapted Comet assay, TBARS assay, and direct quantification of oxidative DNA adducts by GC/MS). Importantly, we will directly test whether pheomelanin functions as a pro-carcinogen by an HPRT-based forward mutagenesis approach in primary melanocytes. Finally, we will directly test the ability of forskolin, an activator of adenylyl cyclase, to bypass defective Mc1r function to enhance recovery from UV-mediated DNA damage and to rescue UV protection in melanocytes and in whole skin. Together, these studies will lay the foundation for future translational studies designed to develop novel small molecule-based approaches for UV and cancer protection to prevent many cases of melanoma.